Genome editing with split Cas9 expressed from two vectors

ABSTRACT

The present invention relates to a method for regulating gene expression, comprising introducing into a cell each of a recombinant vector which expresses a first domain comprising N-terminus of a Cas9 protein, and a recombinant vector which expresses a second domain comprising C-terminus of a Cas9 protein, a composition comprising the recombinant vectors, a kit for regulating gene expression, and a method for intracellular production of Cas9 protein. Moreover, the present invention relates to a transformed cell introduced with a viral vector which packages the first domain, and a viral vector which packages the second domain, and to a composition comprising a virus produced therefrom.

TECHNICAL FIELD

The present invention relates to a method for regulating geneexpression, comprising introducing into a cell each of a recombinantvector which expresses a first domain comprising the N-terminus of Cas9protein, and a recombinant vector which expresses a second domaincomprising the C-terminus of Cas9 protein, a composition comprising therecombinant vectors, a kit for regulating gene expression, and a methodfor intracellular production of Cas9 protein.

Moreover, the present invention relates to a transformed cell introducedwith a viral vector which packages the first domain, and a viral vectorwhich packages the second domain, and to a composition comprising avirus produced therefrom.

BACKGROUND ART

As a tool which is currently being widely used in genetic engineering,restriction enzymes are one of the most important tools in currentmolecular biology research. However, as the need arose for restrictionenzymes which are useful for handling genome-sized DNA and which act asa “rare cutter” capable of recognizing and cleaving a DNA nucleotidesequence having a length of 9 bp or more, various attempts have beenmade.

As a part of such attempts, artificial nucleases, such as meganuclease,zinc-finger nucleases (ZFNs) and TAL-effector nucleases (TALENs), weredeveloped which are tools capable of inducing mutations of endogenousgenes in cells and microorganisms, target gene insertions, andchromosomal rearrangements. These artificial nucleases can beeffectively used as a potent and versatile tool in various fields,including the genetic engineering field, the biotechnology field and themedical field. Recent development of RGENs (RNA-guided engineerednucleases), which are third-generation programmable nucleases using theCRISPR/Cas system known as a microbial immune system, has lead to newdiscovery and innovation in all areas of the biotechnology field (Kim,H. et al., Nat Rev Genet, 2014, 15: 321-334).

The artificial nucleases as described above recognize specific targetnucleotide sequences in cells to induce DNA double strand breaks (DSBs).The induced intracellular DSBs can be repaired by the cell's endogenousDNA repair mechanisms (homologous recombination (HR) and nonhomologousend joining (NHEJ)), in which target-specific mutations and geneticmodifications occur. When a homologous DNA donor is not present ineukaryotic cells and organisms, the DSBs induced by nucleases can bemainly repaired by the NHEJ mechanism rather than the HR mechanism.HR-mediated mutations occur while the sequence in HR donor DNA isexactly copied, but NHEJ-mediated mutations randomly occur. Because NHEJis an error-prone repair mechanism, small insertion/deletion mutations(indel mutations) may occur in regions in which DSBs occurred. Suchmutations induce frame-shift mutations to cause gene mutations.

In particular, the Cas9 protein of the CRISPR/Cas system is a usefultool in designing genetic modifications in eukaryotic cells andorganisms. However, the size of the gene encoding the Cas9 protein islarge, and for this reason, when the Cas9 protein is to be inserted intoa viral vector for intracellular delivery, there is a problem in thatthe efficiency of virus production and the efficiency of intracellulardelivery are low due to the limited packaging of the viral vector. Thus,there is a need for studies focused on expressing the Cas9 protein by aviral vector.

DISCLOSURE OF INVENTION Technical Problem

The present inventors have made extensive efforts to overcome thelimited packaging of a viral vector and to develop a system capable ofexpressing the Cas9 protein by a viral vector. As a result, the presentinventors have divided the Cas9 protein into two domains which can bepackaged into viral vectors, and have constructed recombinant vectorscapable of expressing each of the domains. Furthermore, the presentinventors have found that, when the recombinant vectors are introducedinto a cell, the domains are fused to each other to exhibit Indel(insertion or deletion) effects on genomic target DNA, therebycompleting the present invention.

Technical Solution

It is an object of the present invention to provide a method forregulating gene expression, comprising introducing into a cell each of arecombinant vector, which expresses a first domain comprising N-terminusof a Cas9 protein, and a recombinant vector which expresses a seconddomain comprising C-terminus of a Cas9 protein.

Another object of the present invention is to provide a compositioncomprising a recombinant vector which expresses a first domaincomprising N-terminus of a Cas9 protein, and a recombinant vector whichexpresses a second domain comprising C-terminus of a Cas9 protein.

Still another object of the present invention is to provide a kit forregulating gene expression, comprising the above-described composition.

Yet another object of the present invention is to provide a transformedcell introduced with a viral vector which packages a first domaincomprising N-terminus of a Cas9 protein, and a viral vector whichpackages a second domain comprising C-terminus of a Cas9 protein.

A further object of the present invention is to provide a compositioncomprising a culture or cell lysate of the above-described transformedcell.

A still further object of the present invention is to provide a methodfor intracellular production of Cas9 protein, comprising introducinginto a cell each of a recombinant vector, which expresses a first domaincomprising N-terminus of a Cas9 protein, and a recombinant vector whichexpresses a second domain comprising C-terminus of a Cas9 protein.

Advantageous Effects

The present invention could improve the target specificity of Cas9protein, and also enables to apply the Cas9 protein to the viral vectorso that it can be useful for regulation of gene expression using theCas9 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing a process in which recombinantvectors comprising the first domain and second domain of the Cas9protein, respectively, are constructed, and then introduced andexpressed in a cell, whereby these domains are fused to each other inthe cell to form a full-length Cas9 protein (referred to as Split-Cas9).

FIG. 2 shows the T7 endonuclease 1 (T7E1) mutation detection assayresults indicating that Split-Cas9 protein is formed in a cell and actstogether with sgRNA to induce Indel in all of HPRT, DMD and CCR5 genes(1) Split-Cas9+sgRNA, 2) second domain of the Cas9+sgRNA, 3) firstdomain of the Cas9+sgRNA, 4) mock).

FIG. 3 shows the results of next-generation sequencing performed toanalyze the efficiency of mutagenesis of genes target by the Split-Cas9protein.

FIG. 4 shows the results of analyzing the specificities of theSplit-Cas9 protein for target genes. Specifically, FIG. 4 shows theresults of next-generation sequencing performed to analyze theefficiency of mutagenesis at an on-target site and off-target sites inHela cells (FIG. 4a ) and Hep1 cells (FIG. 4b ). Specificity wasanalyzed by the specificity ratio obtained by dividing the efficiency ofmutagenesis at the on-target site by the efficiency of mutagenesis ateach of four off-target sites.

FIG. 5 shows a process of constructing split-Cas9 delivery vectors andthe results of examining the function of the vectors. Specifically,FIGS. 5(a) and 5(b) schematically show construction of adeno-associatedvirus vectors for delivering split-Cas9. U6 promoter, sgRNA, EFSpromoter, a first domain, and a splicing donor were sequentiallyinserted into an adeno-associated virus vector, thereby constructing avirus vector that packages the first domain. In addition, anadeno-associated virus vector was constructed which includes a splicingacceptor, a second domain or which is capable of packaging the seconddomain together with U6 promoter and sgRNA. FIG. 5(c) shows the resultsobtained by co-infecting Hela cells with 10, 50 and 100 MOI(multiplicity of infectivity) of an adeno-associated virus that packagesU6 promoter, sgRNA, EFS promoter and a first domain and a virus thatpackages a second domain, and after 5, 7 and 10 days, analyzinginduction of mutation in the DMD exon 51 by next-generation sequencing.

BEST MODE FOR CARRYING OUT THE INVENTION

To achieve the above objects, one embodiment of the present inventionprovides a method for regulating gene expression, comprising introducinginto a cell each of a recombinant vector, which expresses a first domaincomprising N-terminus of a Cas9 protein, and a recombinant vector whichexpresses a second domain comprising C-terminus of a Cas9 protein.

As used herein, the term “regulating gene expression” refers to all actsto increase or decrease the expression of the gene. In particular, forthe purpose of the present invention, the regulation of gene expressionmay be performed by Cas9 protein. Specifically, any methods ofincreasing or decreasing the gene expression using Cas9 proteins can beincluded within the scope of the invention without any limitations. Forexample, the regulation of gene expression might refer to genomeediting, increasing gene expression, or decreasing gene expression.

As used herein, the term “genome editing” refers to a technique capableof introducing a targeted mutation into the nucleotide sequence of agene in animal and plant cells, including human cells, and refers toknock-out or knock-in a specific gene, or introducing a mutation into anon-coding DNA sequence that does not produce protein. In addition,genome editing enables deletion, duplication, inversion, replacement orrearrangement of genomic DNA.

As used herein, the term “deletion” refers to a mutation caused bydeletion of a portion of a chromosome or a portion of DNA nucleotides.

As used herein, the term “duplication” means that two or more identicalgenes are present in the genome.

As used herein, the term “inversion” means that a portion of the genomeis arranged inversely relative to the original genome.

As used herein, the term “replacement” means that one nucleotidesequence is replaced by another nucleotide sequence (that is,replacement of a sequence with information), and does not necessarilymean only that one polynucleotide is chemically or physically replacedby another polynucleotide.

As used herein, the term “rearrangement” refers to a structural changeleading to a change in the positions and sequence of a chromosomal gene,and also includes insertion of transposable elements such astransposons. In addition, the term may include the conversion of geneticinformation by nucleotide rearrangement in DNA molecules.

As used herein, the term “Cas9 protein” refers to the major proteinelement of the CRISPR/Cas9 system, which forms a complex with crRNA(CRISPR RNA) and tracrRNA (trans-activating crRNA) to form activatedendonuclease or nickase.

Cas9 protein or gene information can be obtained from a known databasesuch as the GenBank of NCBI (National Center for BiotechnologyInformation), but is not limited thereto. For example, the Cas9 proteinmay be encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2, but is not limited thereto, and any Cas9 protein havingtarget-specific nuclease activity together with guide RNA may beincluded in the scope of the present invention. Furthermore, the Cas9protein may be bound with a protein transduction domain. The proteintransduction domain may be poly-arginine or HIV TAT protein, but is notlimited thereto. In addition, those skilled in the art can appreciatethat an additional domain can be suitably bound to the Cas9 proteinaccording to the intended use.

In addition, the Cas9 protein may comprise not only wild-type Cas9, butalso deactivated Cas9 (dCas9), or Cas9 variants such as Cas9 nickase.The deactivated Cas9 may be RFN (RNA-guided FokI nuclease) comprising aFokI nuclease domain bound to dCas9, or may be dCas9 to which atranscription activator or repressor domain is bound. The Cas9 nickasemay be D10A Cas9 or H840A Cas9, but is not limited thereto.

The Cas9 protein of the present invention is not limited in its origin.For example, the Cas9 protein may be derived from Streptococcuspyogenes, Francisella novicida, Streptococcus thermophilus, Legionellapneumophila, Listeria innocua, or Streptococcus mutans. For the purposeof the invention, the Cas9 protein is one which the size of Cas9 proteinis so large that it may not be effectively expressed in the viralvector, but is not limited thereto.

In the present invention, in order to express Cas9 in a viral vector,vectors capable of expressing a portion of Cas9 were constructed.Specifically, the Cas9 protein was divided into domains capable of beingexpressed from viral vectors, and was expressed from each of thevectors. In the present invention, the first domain and second domain ofthe Cas9 protein refer to portions of the Cas9 protein, and thesedomains are expressed from separate vectors to be fused in a cell. Inthe present invention, the Cas9 protein constructed in this manner wasnamed “split-Cas9” (FIG. 1).

Split-Cas9 of the present invention is characterized in that it isconstructed by dividing a conventional Cas9 protein, which is notpackaged into a viral vector or the like due to its large size, intodomains having a packageable size, and these domains do not lose theirfunction in cells even when these are expressed from the respectivevectors.

As used herein, the term “first domain” refers to a domain comprisingthe N-terminus of the original Cas9 protein, cleaved for theabove-described purposes, and the term “second domain” refers to adomain comprising the C-terminus of the original Cas9 protein. In thepresent invention, the term “first domain” or “second domain” is usedinterchangeably with the term “half domain”. Each of the domains is tobe expressed from viral vectors, and thus may have a size ranging from400 bp to 3.7 kbp, which can be packaged in each viral vector.Specifically, in the present invention, the first domain and the seconddomain are fused to each other to form the original full-length of Cas9protein, and thus the entire size of the Cas9 protein minus the size ofthe other domain would be the size of one domain.

In a specific example of the present invention, a first domain having asize of 2.1 kbp and a second domain having a size of 1.9 kbp wereintroduced into a plasmid vector and a viral vector. As a result, it wasshown that split-Cas9 expressed from the vectors could induce Indel aton-target site in a cell.

Furthermore, those skilled in the art can appreciate that a nucleotidesequence having a specific function may be added to the first domain andthe second domain according to the intended use. For example, the firstdomain and the second domain may further comprise an NLS (nuclearlocalization signal) sequence, a tag sequence, a splicing donor/splicingacceptor sequence, or the like. Furthermore, the first domain may beencoded by the nucleotide sequence of SEQ ID NO: 3, and the seconddomain may be encoded by the nucleotide sequence of SEQ ID NO: 5, butthe scope of the present invention is not limited thereto.

As used herein, the term “vector” refers to an expression vector capableof expressing a target protein in suitable host cells and to a geneticconstruct that includes essential regulatory elements to which a geneinsert is operably linked in such a manner as to be expressed.

As used herein, the term “operably linked” means that a nucleic acidexpression control sequence is functionally linked to a nucleic acidsequence encoding the protein of interest so as to execute generalfunctions. The sequence encoding the first domain or second domain ofthe nuclease DNA according to the present invention is operably linkedto a promoter such that expression of the coding sequence is under theinfluence or control of the promoter. The two nucleic acid sequences(the sequence encoding the first domain or second domain of DNA and thesequence of the promoter region at the 5′ terminus of the encodingsequence) are operably linked to each other when the encoding sequenceis transcribed by inducing the promoter action. Furthermore, the linkingbetween the two sequences induces no frame-shift mutation, and the twosequences are operably linked to each other when an expressionregulatory sequence does not impair the ability to control expression ofeach domain. Operable linkage with the recombinant vector can beperformed using a gene recombination technique well known in the art,and site-specific DNA cleavage and ligation can be performed usingenzymes generally known in the art.

In the present invention, the vector may include an expressionregulatory element such as a promoter, an operator, an initiation codon,a stop codon, a polyadenylation signal, or an enhancer, as well as asignal sequence or a reader sequence for membrane targeting andsecretion, and may be variously manufactured so as to be adapted forsome purpose. The promoter of the vector may be constructive orinductive. Furthermore, the expression vector includes a selectivemarker for selecting a host cell containing the vector, and a replicableexpression vector includes a replication origin. The vector may beself-replicating, or may be integrated into the host DNA. The vectorincludes a plasmid vector, a cosmid vector, a viral vector, and thelike. Specifically, the vector may be the viral vector. An example ofthe viral vector may include, but is not limited to, a vector derivedfrom Retrovirus, for example, HIV (Human Immunodeficiency Virus), MLV(Murine Leukemia Virus), ASLV (Avian Sarcoma/Leukosis), SNV (SpleenNecrosis Virus), RSV (Rous Sarcoma Virus), MMTV (Mouse Mammary TumorVirus), Adenovirus, Adeno-associated virus, Herpes simplex virus, etc.

In the present invention, “introducing into a cell” may use any methodsknown in the art, and a foreign DNA may be introduced into cells bytransfection or transduction. The transfection may be performed byvarious methods known in the art, including calcium phosphate-DNAcoprecipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome-mediatedtransfection, liposome fusion, lipofection and protoplast fusion.

In one example of the present invention, each recombinant vectorencoding each of the first domain and second domain of the Cas9 proteinwas constructed (FIG. 1), and then introduced and expressed in a cell.As a result, it was shown that the expressed domains were fused to eachother in the cell to function as a full-length Cas9 protein.Specifically, it was shown that the first domain and the second domain,which are half domains, were expressed from the recombinant vectors, andthen fused to each other to form a Cas9 form, and the formed Cas9protein acted together with sgRNA to induce Indel (insertion ordeletion) in all target genes (FIGS. 2 and 3).

In another example of the present invention, the target specificities ofthe split-Cas9 protein in Hela cells and Hep1 cells were examined, andas a result, it was shown that the target specificity of the split-Cas9protein was 80 to 220-fold higher than the specificity of wild-type Cas9(FIG. 4). This suggests that when split-Cas9 of the present invention isexpressed in a cell, it can act at a desired on-target sites whileminimizing off-target effects.

In still another embodiment of the present invention, split-Cas9 wasexpressed using adeno-associated virus vectors, and cells were infectedwith the produced virus. As a result, it was shown that split-Cas9effectively induced Indel (FIG. 5). Accordingly, it was found that theCas9 protein can also be effectively used through viral vectorscomprising split-Cas9.

Specifically, when the vectors are introduced into cells, asequence-specific guide RNA may additionally be introduced. Morespecifically, each vector and the guide RNA may be introducedsimultaneously, sequentially or in a reversed order.

In the present invention, the “guide RNA” may consist of two RNAs, i.e.,crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA). Alternatively,the guide RNA may be a sgRNA (single-chain RNA) prepared by the fusionof the main parts of crRNA and tracrRNA. In addition, the guide RNA maybe a dual RNA comprising a crRNA and a tracrRNA.

RGENs known as third-generation programmable nucleases may be composedof Cas protein and dual RNA or may be composed of Cas protein and sgRNA.The guide RNA may comprise one or more additional nucleotides at the 5′terminus of sgRNA or crRNA of dual RNA, and may be deliveredintracellularly as a RNA or a DNA encoding the RNA.

Another embodiment of the present invention provides a compositioncomprising a recombinant vector which expresses a first domaincomprising N-terminus of a Cas9 protein, and a recombinant vector whichexpresses a second domain comprising C-terminus of a Cas9 protein. Thecomposition may be introduced into cells to regulate expression of adesired gene. The composition may further comprise a sequence-specificguide RNA. The Cas9 protein and the recombinant vector are the samethose as described above.

In an example of the present invention, a recombinant vector, whichexpresses the first domain, and a recombinant vector which expresses thesecond domain, were introduced into cells. The reason is to deliver theCas9 protein, which has a size making the Cas9 protein be difficult topackage into a vector, and to express the delivered protein moreefficiently. The use of a composition comprising each of the recombinantvector enables the Cas9 protein to be more easily expressed in cells.The composition may comprise, in addition to the recombinant vectorexpressing the first domain and the recombinant vector expressing thesecond domain, a medium composition capable of maintaining cells or asubstance required to introduce the recombinant vectors into cells.

Still another embodiment of the present invention provides a kit forregulating gene expression, comprising a recombinant vector whichexpresses a first domain of Cas9 protein, and a recombinant vector whichexpresses a second domain of Cas9 protein. Specifically, the kit mayfurther comprise a sequence-specific guide RNA.

Moreover, the kit according to the present invention may comprise notonly a substance that induces or promotes expression of the recombinantvectors or a medium composition capable of maintaining cells, but also acomposition capable of facilitating the construction or intracellularintroduction of the recombinant vectors and a manual for theconstruction or intracellular introduction of the recombinant vectors.

Yet another embodiment of the present invention provides a transformedcell, which is introduced with a viral vector packaging a first domainof the Cas9 protein, and a viral vector packaging a second domain of theCas9 protein.

As used herein, the term “transformed cell” means a cell obtained byintroducing a desired polynucleotide into host cells. Transformation maybe accomplished by the “introduction” method and can be performed byselecting suitable standard techniques according to host cells, as isknown in the art.

It is to be understood that the host cell refers to eukaryotic orprokaryotic cell into which one or more DNAs or vectors are introduced,and refers not only to the particular subject cell but also to theprogeny or potential progeny thereof. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. In the present invention, the transformed cell is a cellintroduced with viral vectors encoding each of the half domains, and avirus that packages the nucleotide sequence encoding each domain of Cas9can be obtained from the transformed cell. Specifically, the virus canbe obtained from a culture or a lysate of the transformed cell.

Examples of the cell include, but are not limited to, prokaryotic cellssuch as E. coli, eukaryotic cells such as yeast, fungi, protozoa, higherplants or insects, mammalian cells such as CHO, HeLa, HEK293 or COS-1,etc.

In addition, the present invention may be applied to all human cells,including somatic cells, germ cells, induced pluripotent stem cells, andadult stem cells.

The somatic cells refer to all cells other than germ cells, which can beobtained from embryos and children and adult bodies, and may alsoinclude genetically modified cells derived therefrom. In addition, theadult stem cells may include not only all adult stem cells obtainablefrom human embryos, neonates and adult bodies, but also extraembryonicstem cells, including cord blood stem cells, placenta stem cells,Wharton's jelly stem cells, amniotic fluid stem cells, and amnioticepithelial cells, as well as genetically modified cells derivedtherefrom.

In addition, the cells may also be cultured cells (in vitro), graft andprimary cultures (in vitro and ex vivo), or in vivo cells, and are notparticularly limited as long as they are cells that are generally usedin the art.

In another embodiment of the present invention, there is provided acomposition comprising a culture or cell lysate of the transformed cell.The transformed cell and the culture and lysate thereof are as describedabove. The composition comprises a virus that packages the nucleotidesequence encoding each of the domains, and thus may be used to regulategene expression.

According to the present invention, the limitation in packaging of theCas9 protein by a vector is overcome, and the efficiency ofintracellular delivery of the Cas9 protein is increased by constructingrecombinant vectors that individually express the two cleaved domains ofthe Cas9 protein and delivering the constructed recombinant vectors tobe expressed in cells. Thus, the inventive principle developed by thepresent inventors may be applied regardless of the types of cells or thetypes of Cas9 protein to increase the efficiency of intracellulardelivery of Cas9 protein to thereby efficiently regulate geneexpression.

MODE FOR INVENTION

Hereinafter, the present invention will be described in detail withreference to examples. It will be obvious to a person having ordinaryskill in the art that these examples are for illustrative purposes onlyand are not to be construed to limit the scope of the present invention.

EXAMPLE 1 Construction of Recombinant Vectors Expressing Each of Firstand Second Domains of Cas9 Protein

The middle portion of a disordered linker (SEQ ID NO: 9; agcggccagggc;the sequence encoding SGQG amino acids) present in the middle portion ofwild-type (WT) Cas9 (CRISPR associated protein 9) protein (SEQ ID NO: 2)was cleaved, thereby constructing two half domains in which SG aminoacids and QG amino acids were linked to the first domain and the seconddomain, respectively.

Each of the half domains was configured such that independent domainthereof could be induced by a CMV promoter. A stop codon was insertedinto the cleaved 3′-end of the first domain by PCR cloning such thatexpression could be completed, and a start codon was linked to thecleaved 5′-end region of the second domain such that expression could beinitiated. A HA tag and a NLS (nuclear localization signal) weresequentially inserted downstream of the start codon in the 5′-end regionof the first domain, a NLS region and a HA tag were sequentiallyinserted between the 3′-end and stop codon of the second domain, suchthat protein expression could be measured by nuclear localization andthe HA antibody.

EXAMPLE 2 Examination of Intracellular Introduction of RecombinantVectors Expressing Each Half Domain and sgRNA and Knock Out of TargetGenes

The recombinant vectors expressing each half domain, constructed inExample 1, plasmids expressing sgRNA (single guide RNA) for each ofCCR5, HPRT and DMD genes, were delivered into cells by transfectionusing lipofectamin.

As a target sequence for allele knockout of the CCR5 gene, a conversedsequence commonly present in all humans was used, and the5′-TGACATCAATTATTATACATCGG-3′ sequence (SEQ ID NO: 11) present in CCR5exon 2 was targeted.

Furthermore, as a target sequence for allele knockout of the HPRT gene,the 5′-GCCCCCCTTGAGCACACAGAGGG-3′ sequence (SEQ ID NO: 12) present inDMD exon 51 was targeted.

In addition, as a target sequence for allele knockout of the DMD gene,the 5′-TCCTACTCAGACTGTTACTCTGG-3′ sequence (SEQ ID NO: 13) present inDMD exon 51 was targeted.

Next, genomic DNAs were extracted from the Hela cells, and then thetarget sequence region in each of the HPRT, DMD and CCR5 genes wasamplified by PCR.

Next, whether Indel (insertion or deletion) was induced was analyzed byT7E1 (T7 endonuclease I) mutation detection assay, and the results ofagarose gel analysis are shown in FIG. 2. The T7E1 assay was performedaccording to a known method. In brief, genomic DNAs were isolated usingthe DNeasy Blood & Tissue Kit (G-DEX IIc Genomic extraction kit)according to the manufacturer's instruction.

As can be seen in FIG. 2, each of the half domains was expressed fromthe intracellularly introduced recombinant vectors (constructed inExample 1), and then the expressed half domains were fused to each otherto form Cas9 protein (named “Split-Cas9”) which then acted together withsgRNA to induce Indel in all the HPRT, DMD and CCR5 genes.

EXAMPLE 3 Analysis of Knockout Efficiency of Target Genes by Split-Cas9and Target Gene-Specific sgRNA

In order to analyze the knockout efficiency of target genes, targetsequence regions were amplified by PCR, and then the target sequenceswere analyzed by a next-generation assay. The results of the analysisindicated that the Indel frequency was 27.1% in the HPRT gene, 23.75% inthe DMD gene, and 20.27% in the CCR5 gene. In a control group, only onehalf domain for the first or second domain was introduced and expressedin cells, and in this case, no Indel appeared (FIG. 3).

From the results as described above, it could be seen that, when therecombinant vectors expressing each half domain of Cas9 were introducedinto cells, the half domains were expressed normally and then fused toeach other to form a full-length Cas9 protein, indicating that the halfdomains can act together with sgRNA to exhibit Indel effects on targetgenes. Cas9 has reduced intracellular delivery efficiency due to thesize of a Cas9 expression cassette, which is larger than a size capableof being packaged in a viral vector. According to the present invention,the first and second domains of Cas9 are introduced individually intocells so that they can be expressed in the cells and then fused to eachother to exhibit their function, thereby solving the problem associatedwith the packaging of the Cas9 protein into a vector.

EXAMPLE 4 Analysis of Target Sequence Cleavage Specificity of Split-Cas9

In order to analyze the off-target effect of target genes, cells weretreated with each of split-Cas9 and wild-type Cas9 plasmids, and after 3days, a similar sequence region having a sequence mismatch with thetarget sequence of the HBB gene was amplified by PCR. Next, the targetsequence was analyzed by a next-generation assay.

When on-target efficiency in Hela cells was divided by off-targetefficiency, it was shown that the specificity of split-Cas9 was up to220-fold higher than the specificity of wild-type Cas9 (FIG. 4a ). Inaddition, it was shown that the specificity of split-Cas9 in Hep1 cellswas up to 80-fold higher than the specificity of wild-type Cas9 (FIG. 4b).

EXAMPLE 5 Analysis of Target Sequence Cleavage Specificities byAdeno-Associated Virus Expressing Split-Cas9

In order to examine whether Split-Cas9 effectively acts even when it isdelivered using viral vectors, each of the first and second domains wascloned into adeno-associated virus vector plasmids (FIGS. 5a and 5b ).

A splicing donor was linked to the C-terminal region of the firstdomain, and a splicing acceptor was linked to the N-terminal region ofthe second domain. Viruses that package each of the half domains wereproduced, recovered and delivered intracellularly, and then the cleavagerate of the target sequence region was analyzed.

As a result, it could be seen that the half domains were fused to eachother to form a full-length Cas9 protein to thereby exhibit genecleavage effects. Meanwhile, Hela cells were infected with 10, 50 and100 MOI (multiplicity of infectivity) of adeno-associated virus fordelivering Split-Cas9, and after 5, 7 and 10 days, the target sequencecleavage rate was analyzed. As a result, it could be seen that a targetsequence cleavage effect of about 5% appeared (FIG. 5c ). This suggeststhat split-Cas9 of the present invention effectively acts even when itis delivered using viral vectors.

From the foregoing, it will be understood by those skilled in the art towhich the present invention pertains that the present invention can becarried out in other concrete embodiments without changing the technicalspirit or essential feature thereof. In this regard, it should beunderstood that the aforementioned examples are of illustrative in allaspects but not is limited. The scope of the present invention should beconstrued to include the meaning and scope of the appended claims, andall the alterations and modified forms which are derived from theequivalent concept thereof, rather than the detailed description.

The invention claimed is:
 1. A method for introducing a targetedmutation into a genome, comprising introducing into an isolated cell (i)a recombinant vector, which expresses a first domain comprising theN-terminus of a Cas9 protein, and (ii) a recombinant vector whichexpresses a second domain comprising the C-terminus of a Cas9 protein,wherein the first domain is encoded by the nucleotide sequence of SEQ IDNO: 3, and the second domain is encoded by the nucleotide sequence ofSEQ ID NO: 5, wherein the first domain and the second domain areconstructed by cleaving a middle portion of the sequence of SEQ IDNO.:10 in the Cas9 protein, in which SG amino acids and QG amino acidsare respectively linked to the first domain and the second domain. 2.The method of claim 1, wherein the Cas9 protein is derived from any oneselected from the group consisting of Streptococcus pyogenes,Francisella novicida, Streptococcus thermophilus, Legionellapneumophila, Listeria innocua, and Streptococcus mutans.
 3. The methodof claim 1, wherein the recombinant vector is a plasmid vector, a cosmidvector, or a viral vector.
 4. The method of claim 3, wherein the viralvector is selected from the group consisting of a retrovirus vector, anadenovirus vector, an adeno-associated virus vector, and a herpessimplex virus vector.
 5. The method of claim 1, further comprisingfusing the first domain and the second domain, which are expressed fromeach of the introduced recombinant vectors, to form the Cas9 protein. 6.The method of claim 1, wherein the first domain and the second domaineach comprises an NLS (nuclear localization signal) sequence, anhemagglutinin (HA) tag sequence, a splicing donor sequence, a splicingacceptor sequence, or a combination thereof.
 7. The method of claim 1,wherein a sequence-specific guide RNA is introduced into the cell. 8.The method of claim 7, wherein introducing each vector and the guide RNAis performed in a simultaneous or sequential manner.